Cynthia Weinig and Matthew Rubin Phenotypic Variation in Circadian Near Isogenic Lines of Arabidopsis thaliana Sarah Cheeney Advisors: Cynthia Weinig and Matthew Rubin

Circadian Rhythms The Earth rotates on its axis once every 24 hours Providing predictable cycles of light and temperature Most organisms exhibit circadian rhythms Circadian rhythms are endogenous repeating rhythms Circadian rhythms are “set” by external cues Many physiological and behavioral characters are controlled by circadian rhythms Best example in plants is photosynthesis 2

Arabidopsis thaliana: The Real-Live Plant Member of the mustard family Related to cabbage, cauliflower, kohlrabi, broccoli, and kale Useful for environmental studies: Primarily self-fertilizing Naturally highly inbred families Grow replicates of each genotype in each environment. Short-lived annual Fitness estimated from fruit number

Research Questions Do previously characterized circadian NILs show patterns in other non-circadian traits? Can physiological parameters be measured at both the whole plant and leaf level? Are any of the measured traits adaptive?

Recombinant Inbred Lines Parent 1 Parent 2 X Each RIL is a novel combination of parental alleles F1 (8 more generations)

Temperature Compensation Temperature compensation in Ler, Col, Cvi, and the CvL and CoL RILs. (A) Mean leaf-movement period plotted against temperature for the Ler (open circles), Col (solid triangles), and Cvi (solid circles) accessions. Edwards K D et al. Genetics 2005;170:387-400 6

QTL Mapping QTL mapping tests for an association between a genetic marker state and phenotype marker M M m M m m m m M QTL Q Q q Q q q q q Q M = Marker allele 1 m = Marker allele 2 Q = Allele for branching q = Allele for fewer branches

QTL Mapping Results Genetic mapping of circadian period QTL in the CvL and CoL RILs. QTL likelihood (LOD) maps across Arabidopsis chromosomes 1 and 5 in the CvL (A) and CoL (B) RILs. Chromosome numbers are indicated in the upper right corner of each graph. Names on the x-axis correspond to molecular markers. QTL were mapped independently at 12° (open diamonds), 22° (shaded diamonds), and 27° (solid diamonds). Dashed line represents 2.7 LOD significance threshold of P < 0.05 (as calculated from Van Ooijen 1999). Solid boxes on x-axis span the 2-LOD support interval of mapped QTL. Putative QTL designations are indicated in italics. Selected markers used as cofactors in mapping are identified above the x-axis with diamonds shown as open (12°), shaded (22°), and solid (27°) according to temperature. Edwards K D et al. Genetics 2005;170:387-400 8

NILs test the effect of a single introgression PerCv1a, PerCv1b, and PerCv5d NILs. Mean leaf-movement period of Ler is compared to NILs for PerCv1a and PerCv1b (A and B) and for PerCv5d (D and E) at 12°, 22°, and 27°. See insets for line identification. Error bars represent SEM of period estimates. NIL genotypes across the five Arabidopsis chromosomes are shown to the right of each graph. Open regions represent the Ler genotype and solid regions represent the Cvi genotype. Detailed view of NIL genotypes around PerCv1a and PerCv1b (C) and PerCv5d (F) regions shows the position of mapping markers (solid diamonds) and candidate genes (open diamonds). Horizontal lines correspond to mapping markers and solid (Cvi) and open (Ler) regions of vertical bars represent the genotype of NILs at the markers. Breakpoints are estimated as the midpoint between mapping markers. Edwards K D et al. Genetics 2005;170:387-400 9

Greenhouse Project Genotypes: Cvi x Ler NILs 16 replicates of each NIL Grew at warm temperature (from Edwards et al) As a result we have NILs that are either short, WT, or long period Cold-treated seeds for 4 days Measure Physiology traits Days to Germination Days to Flowering Leaf number Leaf size

IRGA (infra-red gas analyzer) gas exchange system Parameters measured or calculated: photosynthetic carbon assimilation (Photo), stomatal conductance (Cond), intercellular CO2 concentration (Ci), fluorescence (Fs), leaf transpiration, vapor pressure deficit calculated from measure leaf temperature (VpdL), measured air temperature (Tair), measured leaf temperature (Tleaf),

Days to Germination and Bolting Advanced in Short and Long Period NILs

Short Period NILs Have Reduced Size and Leaf Number

Net photosynthesis and quantum yield tend to be higher in WT plants

Difficulties in Physiological Measurements Whole plant IRGA requires plants to be at an earlier developmental stage than other studies making it difficult to compare across experiments Soil respiration is confounded with plant measurements Overcome by “capping soil” with plastic or clay

Circadian Clock is Adaptive Earth rotates every 24 hours providing predictable cycles of light and temperature WT and WT-like NILs have increased leaf number and size. These two traits are often positively correlated with fruit set. WT plants tended towards higher net photosythesis and more effiecient photosytem II activity.

Future Research Increase replication Lead to better sampling of NILs and period classes

Acknowledgements Funding NSF EPSCoR Research Fellowships for Undergraduates Thanks to: Advisor: Cynthia Weinig and Matthew Rubin Planting/Data Collection : Kyle Anderson, Meredith Pratt and Yulia Yarkhunova